Comparing the effects of atrazine and an environmentally relevant mixture on estrogen-responsive gene expression in the northern leopard frog and the fathead minnow.

In Nebraska, fish are exposed to herbicides in agricultural runoff. The study objectives were to determine 1) if fathead minnows and northern leopard frogs exposed to atrazine experience alterations in gene expression, and 2) whether these changes are elicited by a simulated herbicide mixture. Following a 7-d exposure to atrazine, female minnows were defeminized, whereas male frogs were feminized. The mixture did not elicit statistically significant effects in either species. Environ Toxicol Chem 2018;37:1182-1188. © 2018 SETAC.

A new species of leopard frog (Anura: Ranidae) from the urban northeastern US.

Past confusion about leopard frog (genus Rana) species composition in the Tri-State area of the US that includes New York (NY), New Jersey (NJ), and Connecticut (CT) has hindered conservation and management efforts, especially where populations are declining or imperiled. We use nuclear and mitochondrial genetic data to clarify the identification and distribution of leopard frog species in this region. We focus on four problematic frog populations of uncertain species affiliation in northern NJ, southeastern mainland NY, and Staten Island to test the following hypotheses: (1) they are conspecific with Rana sphenocephala or R. pipiens, (2) they are hybrids between R. sphenocephala and R. pipiens, or (3) they represent one or more previously undescribed cryptic taxa. Bayesian phylogenetic and cluster analyses revealed that the four unknown populations collectively form a novel genetic lineage, which represents a previously undescribed cryptic leopard frog species, Rana sp. nov. Statistical support for R. sp. nov. was strong in both the Bayesian (pp=1.0) and maximum-likelihood (bootstrap=99) phylogenetic analyses as well as the Structure cluster analyses. While our data support recognition of R. sp. nov. as a novel species, we recommend further study including fine-scaled sampling and ecological, behavioral, call, and morphological analyses before it is formally described.

Developmental profiles and thyroid hormone regulation of brain transcripts in frogs: a species comparison with emphasis on Physalaemus pustulosus.

In amphibians, thyroid hormones (THs) are considered key regulators of brain remodeling during metamorphosis, while sex steroids (estrogens and androgens) control sexual differentiation and gonadal development. However, these two endocrine axes can interact during tadpole brain development. Previously, we demonstrated that THs affect sex steroid-related gene expression in the developing brain of Silurana tropicalis and Rana pipiens; however, the gene expression changes differed between species. We chose to study a third anuran species, Physalaemus pustulosus, to test new hypotheses about the role of THs in the regulation of brain gene expression. We first established developmental transcript profiles of TH- and sex steroid-related genes in the brain of P. pustulosus. Then, following the same protocols as in our previous studies, we investigated triiodothyronine (T3) regulation of brain transcripts in premetamorphic P. pustulosus and then compared the results with our previous two studies. In the case of TH-related genes, TH receptor beta (trbeta) and deiodinase type 3 (dio3), mRNA developmental profiles were similar in the three species and with respect to other species in the published literature. However, the profiles of TH receptor alpha (tralpha) and deiodinase type 2 (dio2) mRNA revealed differences between anuran species. Among the three anurans we have studied, the direction of the T3 regulation of TH-related genes was overall similar, but the magnitude of gene expression change differed depending on the rate of metamorphosis in a given species. For the sex steroid-related genes, each species exhibited similar developmental profiles but differed in their response to T3. In P. pustulosus, T3 reduced the expression of aromatase (cyp19) while increasing mRNA levels of androgen and estrogen receptors. These results are similar to previous research in R. pipiens but differ from data for S. tropicalis, for which we found an increase in androgen synthesis enzymes but no effect on cyp19. Together, we propose that T3 has the potential to induce the brain androgen system in anurans. This could be achieved by increasing androgen synthesis enzymes (S. tropicalis) or by decreasing estrogen synthesis (due to a decrease in cyp19 in P. pustulosus and R. pipiens). In conclusion, we demonstrated that mechanisms of hormone interactions differ between anuran species, but in all cases T3 appears to affect the balance of sex steroids in the brain, stimulating the androgen system. We have shown that the regulation of sex steroid-related genes by T3 is more similar among closely related species than species with similar reproductive and developmental characteristics.

Phylogeographic analyses of the southern leopard frog: the impact of geography and climate on the distribution of genetic lineages vs. subspecies.

The southeastern United States is a major phylogeographic break hotspot for amphibians, but the processes underlying this hotspot remain to be explicitly tested. We test the correlation of genetic lineages with subspecies breaks in the southeastern United States and the association of such breaks with climate, using Rana sphenocephala as a case study, and place our results in the broader context of the Alabama-Appalachian suture zone (AL-Appalachian SZ). We use genetic and ecological methods to (i) determine whether genetic lineages are coincident with the AL-Appalachian SZ or the subspecies and (ii) test the correlation of major climatic breaks with genetic structure and morphological variation in R. sphenocephala. Bayesian phylogenetic analyses of the ND1 mtDNA gene and microsatellite cluster analyses revealed two distinct lineages with over 4% sequence divergence. The geographic distributions of the two lineages are concordant with the AL-Appalachian SZ but do not correspond to the ranges of the subspecies based on morphology. Mantel tests revealed that isolation by distance and historical barriers to gene flow, rather than climate, are the major drivers of genetic divergence at neutral loci. Examination of climate breaks across the Southeast revealed a pattern incongruent with suture zone hotspots, suggesting that phylogenetic structure has been driven primarily by historical factors, such as isolation, the Appalachian Mountains and the Apalachicola/Chattahoochee/Flint River Basin. However, climate breaks are consistent with the geographic distribution of the subspecies of R. sphenocephala, suggesting that environmental pressures may be driving divergence in morphological traits that outpaces molecular evolution.

A revised leopard frog phylogeny allows a more detailed examination of adaptive evolution at ranatuerin-2 antimicrobial peptide loci.

Ranatuerins are antimicrobial peptides of the innate immune system found in ranid frogs. We previously presented evidence that a positive selective sweep had fixed a single allele at the Ranatuerin2 locus in the northern leopard frog (Rana pipiens). In this paper, we further investigate the evolutionary history of ranatuerins as follows. First, we sequenced Ranatuerin2 in additional individuals of R. pipiens and related frog species and compared diversity and divergence at these sequences with that at four putatively neutrally evolving loci. Second, we asked whether the evolutionary patterns observed at Ranatuerin2 were typical for ranatuerin loci by sequencing our samples at a paralogous locus, Ranatuerin2b, and performing the same neutrality tests. Ranatuerin2b also showed strong and significant evidence of at least one selective sweep. Third, we used the neutral loci to independently resolve conflicting hypotheses about phylogenetic relationships among our study species. Both the neutral loci and the ranatuerin loci supported an older phylogeny inferred from allozyme data and strongly rejected a more recent phylogeny inferred from mitochondrial DNA. Finally, in order to test whether the sweep was driven by the evolution of substantially new peptide function, we used the phylogeny to reconstruct the hypothetical Ranatuerin2 peptide that existed before the sweep. We synthesized this peptide and tested its activity and that of the extant peptide against six bacterial pathogens of frogs. We observed antibacterial activity but found no significant functional differences between the two peptides.

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization.

Onconase, an RNAse extracted from embryos of the Northern leopard frog (Rana pipiens), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an alpha-mating factor (alpha-MF)-prepro secretion signal, and an alpha-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L(-1) for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.

Phylogenetic relationships of leopard frogs (Rana pipiens complex) from an isolated coastal mountain range in southern Sonora, Mexico.

Mitochondrial DNA sequence data from the control region and 12S rRNA in leopard frogs from the Sierra El Aguaje of southern Sonora, Mexico, together with GenBank sequences, were used to infer taxonomic identity and provide phylogenetic hypotheses for relationships with other members of the Rana pipiens complex. We show that frogs from the Sierra El Aguaje belong to the Rana berlandieri subgroup, or Scurrilirana clade, of the R. pipiens group, and are most closely related to Rana magnaocularis from Nayarit, Mexico. We also provide further evidence that Rana magnaocularis and R. yavapaiensis are close relatives.

Cytonuclear discordance across a leopard frog contact zone.

We conducted a genetic analysis of the extent of hybridization within and outside a contact zone between two North American leopard frogs, Rana blairi and Rana pipiens by comparing distribution patterns of mitochondrial and nuclear haplotypes. The contact zone, located between South Dakota, Nebraska, and Iowa, USA, was previously examined using morphological data in the 1970s, leading to the conclusion that hybridization was rare between R. pipiens and R. blairi. Our genetic analysis of 51 populations (611 samples) shows strong cytonuclear discordance. Mitochondrial-haplotype distribution matches the same pattern as the documented species spatial distributions based on morphology. However, the geographic distribution of the nuclear haplotypes reveals asymmetrical swamping of the R. pipiens nuclear haplotypes by R. blairi haplotypes. Phylogenetic analyses of both mitochondrial and nuclear markers provide strong evidence for the presence of R. blairi-R. pipiens introgression for the nuclear marker. A pattern of mitochondrial isolation with nuclear introgression is extremely unusual, and predicted to occur much less often than the reverse.

Hormone cross-regulation in the tadpole brain: developmental expression profiles and effect of T3 exposure on thyroid hormone- and estrogen-responsive genes in Rana pipiens.

During metamorphosis, the tadpole neuroendocrine brain is a major target for the organisational effects of hormones acting via both endocrine feedback mechanisms and local hormone production. While the receptor-mediated actions of thyroid hormones in brain development have been well described, there is evidence that thyroid hormones could also be an important modulator of estrogen action during metamorphosis. To better understand hormone action and potential cross-regulation between thyroid hormone and estrogen, we examined changes in thyroid hormone receptors (TRalpha and TRbeta) and the estrogen receptor (ERalpha) in the brain of Rana pipiens throughout metamorphosis and in response to 48 h waterborne triiodothyronine (T3) exposure (0.5, 5 and 50 nM). We also measured mRNA levels of iodothyronine deiodinase (D2 and D3) and aromatase, key enzymes responsible for local synthesis and availability of thyroid hormones and estrogen, respectively. A real-time PCR strategy targeting these genes was developed using either a fluorescent dual-labelled probe- or SYBR Green I-based method. TRbeta mRNA levels were increased during development and in response to T3 exposure. Deiodinase (D2 and D3) enzymes were differentially regulated during development, but mRNA levels of both were increased with 50 nM T3 exposure. ERalpha and aromatase mRNA levels significantly increased at metamorphic climax, but whereas estrogen receptor alpha mRNA levels were increased by 50 nM T3, aromatase mRNA levels were decreased. These results (1) demonstrate that the developing amphibian brain is an important site for stage-specific thyroid hormone regulation of nuclear receptors and hormone synthesis enzymes and (2) provide the basis for further studies exploring the physiological and functional significance of the cross-regulation between thyroid status and estrogen-sensitive genes in the brain during amphibian metamorphosis.

Characterization and steroidal regulation of gonadotropin beta subunits in the male leopard frog, Rana pipiens.

In ranid frogs, the secretion of gonadotropins (GtHs), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), is potently regulated by gonadal steroids. To better understand the gonadal regulation of GtHs at the molecular level, we elucidated the full-length cDNA sequences of LH and FSH beta subunits from the leopard frog, Rana pipiens. The cDNAs for LHbeta and FSHbeta were 1084 and 667 bp in size excluding the poly (A) tail, and encoded proteins of 138 and 127 amino acids, respectively. Using reverse-transcription polymerase chain reaction (RT-PCR), the messages for LHbeta and FSHbeta were found in the pituitary, but not in the brain, heart, kidney, or the liver. Semi-quantitative RT-PCR revealed a significant elevation of FSHbeta, but not LHbeta, in mature male R. pipiens 21 days after gonadectomy (GDX). 17beta-estradiol implant for 21 days in GDX male frogs significantly suppressed the levels of both LHbeta and FSHbeta transcripts, whereas 5alpha-dihydrotestosterone implant suppressed only the latter. Together, these results laid the groundwork for investigating gonadal regulation of GtHbeta subunits in a ranid frog. Importantly, these data also revealed differential feedback effects of an androgen and an estrogen upon GtHbeta expression.

An analysis of selection on a colour polymorphism in the northern leopard frog.

In this study, we investigated the role of selection in the maintenance of a dorsal colour polymorphism in natural populations of the northern leopard frog, Rana pipiens. We determined genetic structure both spatially and temporally from a suite of putatively neutral molecular markers and tested whether or not the colour locus exhibited patterns of genetic variation that differed from those of the neutral loci. Spatial genetic structure at the colour locus was indistinguishable from structure at neutral loci [95% confidence intervals of F(ST) (neutral) = (0.07, 0.35), F(ST) (colour locus) = 0.114]. In the temporal analysis, we found that the variance among populations in the change in allele frequency at the colour locus (equal to 0.004) lies within the 95% confidence intervals for the variance among populations in changes in allele frequencies at neutral loci. In light of our inability to show evidence for the selective maintenance of the colour polymorphism, we used computer simulations to infer the power of our spatial and temporal techniques to detect selection. The computer simulations showed that although the strength of selection (s) would need to be relatively strong to have been detected by the temporal approach (s = 0.1-0.4, depending on the model tested), the spatial analysis would have detected all but weak selection (s = 0.01-0.04, depending on the model tested). This study illustrates the importance of using a locus comparison approach to detect evidence for selective maintenance before conducting studies to measure the selective mechanisms maintaining a polymorphism.

Partial nucleotide sequences and expression patterns of frog (Rana pipiens) ephrin-A2 and ephrin-A5 mRNA.

We have generated 362 bp and 547 bp partial sequences for Rana pipiens ephrin-A2 and ephrin-A5 mRNA, respectively. Translation homologies for the comparable segments of cDNA of chicken, mouse and human are 90.8, 86.9 and 84.4% for the ephrin-A2 sequence and 85.7, 85.0 and 85.0% for the ephrin-A5 sequence. Digoxigenin-labeled riboprobes were prepared and applied by means of in situ hybridization to whole-mounts of the brains of mature adults and expression patterns in tadpoles were also explored. The RNA probes revealed similar posterior (high) to anterior (low) expression gradients in the adult tectum, demonstrating that both ephrin-As are expressed in the adult Ranid frog tectum. Only the ephrin-A2 probe was tested on tadpole brain, yielding an appropriately graded expression pattern similar to the adult.

Amelogenin sequence and enamel biomineralization in Rana pipiens.

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui.

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.

Effective population sizes and temporal stability of genetic structure in Rana pipiens, the northern leopard frog.

Although studies of population genetic structure are very common, whether genetic structure is stable over time has been assessed for very few taxa. The question of stability over time is particularly interesting for frogs because it is not clear to what extent frogs exist in dynamic metapopulations with frequent extinction and recolonization, or in stable patches at equilibrium between drift and gene flow. In this study we collected tissue samples from the same five populations of leopard frogs, Rana pipiens, over a 22-30 year time interval (11-15 generations). Genetic structure among the populations was very stable, suggesting that these populations were not undergoing frequent extinction and colonization. We also estimated the effective size of each population from the change in allele frequencies over time. There exist few estimates of effective size for frog populations, but the data available suggest that ranid frogs may have much larger ratios of effective size (Ne) to census size (Nc) than toads (bufonidae). Our results indicate that R. pipiens populations have effective sizes on the order of hundreds to at most a few thousand frogs, and Ne/Nc ratios in the range of 0.1-1.0. These estimates of Ne/Nc are consistent with those estimated for other Rana species. Finally, we compared the results of three temporal methods for estimating Ne. Moment and pseudolikelihood methods that assume a closed population gave the most similar point estimates, although the moment estimates were consistently two to four times larger. Wang and Whitlock's new method that jointly estimates Ne and the rate of immigration into a population (m) gave much smaller estimates of Ne and implausibly large estimates of m. This method requires knowing allele frequencies in the source of immigrants, but was thought to be insensitive to inexact estimates. In our case the method may have failed because we did not know the true source of immigrants for each population. The method may be more sensitive to choice of source frequencies than was previously appreciated, and so should be used with caution if the most likely source of immigrants cannot be identified clearly.

Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase.

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.

Evolutionary history of the northern leopard frog: reconstruction of phylogeny, phylogeography, and historical changes in population demography from mitochondrial DNA.

This study uses a combined methodological approach including phylogenetic, phylogeographic, and demographic analyses to understand the evolutionary history of the northern leopard frog, Rana pipiens. We tested hypotheses concerning how (or if) known geological events and key features of the species biology influenced the contemporary geographic and genetic distribution of R. pipiens. We assayed mitochondrial DNA variation from 389 individuals within 35 populations located throughout the species range. Our a priori expectations for patterns and processes influencing the current genetic structure of R. pipiens were supported by the data. However, our analyses revealed specific aspects of R. pipiens evolutionary history that were unexpected. The phylogenetic analysis indicated that R. pipiens is split into populations containing discrete eastern or western haplotypes, with the Mississippi River and Great Lakes region dividing the geographic ranges. Nested clade analysis indicated that the biological process most often invoked to explain the pattern of haplotype position is restricted gene flow with isolation by distance. Demographic analyses showed evidence of both historical bottlenecks and population expansions. Surprisingly, the genetic evidence indicated that the western haplotypes had significantly reduced levels of genetic diversity relative to the eastern haplotypes and that major range expansions occurred in both regions well before the most recent glacial retreat. This study provides a detailed history of how a widespread terrestrial vertebrate responded to episodic Pleistocene glacial events in North America. Moreover, this study illustrates how complementary methods of data analysis can be used to disentangle recent and ancient effects on the genetic structure of a species.

Historical data refute recent range contraction as cause of low genetic diversity in isolated frog populations.

This study tested whether low genetic diversity in remnant populations of a declining amphibian is best explained by recent bottlenecks or by a history of being peripheral. We compared diversity from eight microsatellite loci in historical and extant populations from the interior and former periphery of the species' range. We found that historic peripheral populations already had reduced levels of genetic variation before the range contraction. Therefore, low diversity in remnants could not be ascribed to recent range contractions. This study shows that a common conservation strategy for rescuing genetically depauperate populations, artificial gene flow, may often be unwarranted and detrimental to evolutionarily important peripheral populations.

The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells.

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were approximately 90% identical to a predicted transposase gene sequence from Xenopus laevis, but contained an unpredicted, approximately 180 bp region encoding the N-terminus of the putative transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the transposase gene was derived. This engineered transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only approximately 50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows approximately 70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.